Introduction The cell cycle is regulated by the activities of cyclin-dependent kinases (Cdks). In mammals, 11 different Cdks have been identified whereas in yeast only one major Cdk drives the cell cycle. Of the 11 mammalian Cdks, Cdk4 and Cdk6 promote entry and progression through G1, Cdk2 functions in entry and progression through S-phase, Cdc2 (Cdk1) regulates M-phase, and the other Cdks function in transcription or have unknown functions. The activities of Cdks are regulated by protein-protein interactions (with cyclins, inhibitors, and assembly factors), protein degradation, transcriptional control, subcellular localization, and multiple phosphorylations. Several functions for Cdk2 have been suggested, including entry into S-phase by Rb phosphorylation, initiation of DNA replication, exit from S-phase, and progression through G2 phase. We are investigating the functions of Cdk2 in an in vivo model system as well as in vitro by screening for inhibitors that prevent Cdk2 activation. Aims - Aim 1: Determination of the functions of Cdk2 and Cdc2 in the mouse model system. - Aim 2: Regulation of CDK6/KSHV-cyclin complexes. - Aim 3: In vivo functions of phospho-threonine 160 in Cdk2 Aim 1: Determination of the functions of Cdk2 and Cdc2 in a mouse model system. We are generating Cdk2 knockout mice to study the functions of Cdk2 in different tissues or at different times in the mouse development. We have generated Cdk2 mice and recently obtained heterozygote animals. These mice will be expanded and crossed to obtain homozygotes Cdk2 mutants. In addition, we also finished a targeting vector for a conditional Cdk2 knockout mouse using the Cre/lox system. This vector will be electroporated into ES cell in the near future. We have started to analyze the expression and functions of Cdk2 in the mouse and in mouse embryo fibroblasts. In situ hybridizations have been done on E9.5 mouse embryos and revealed that Cdk2 and Cdk4 are uniformly expressed. We will expand this analysis using not only wild-type embryos but also Cdk2-/- embryos and probes for Cdc2, Cdk6, cyclin D, cyclin E, cyclin A, cyclin B, p27, p21, p16, etc. Such experiments will further our knowledge of Cdk2 in the mouse and help to evaluate possible phenotypes of the Cdk2-/- mice. We also started to work on the Cdc2 locus in the mouse. We plan to make conditional Cdc2 knockout mice and to knockin Cdk2 into the Cdc2 locus. Currently, we have isolated a BAC to contains the Cdc2 locus and have retrieved a 14 kb piece of DNA including the Cdc2 locus. This will form the basis for our future Cdc2 targeting vectors. Aim 2: Regulation of CDK6/KSHV-cyclin complexes. One step in the activation of Cdks is phosphorylation by the Cdk-activating kinase (CAK). We demonstrated the KSHV-cyclin could activate CDK6 in the absence of CAK phosphorylation in vitro and in vivo. A possible explanation is that CDK6 and/or KSHV-cyclin are phosphorylated at unknown sites. We have tried to identify such phosphorylation sites by sequencing and by mass spectrometry. Both methods have not yet produced to results we were looking for. In the mean time, we made mutations at potential phosphorylation sites. For KSHV-cyclin, we made more than 20 mutants and found in a first analysis that all of them are active. This is quite remarkable and proves that KSHV-cyclin seems to be resistant to inactivation by mutation. We also made mutants of CDK6 and half of them seem to be inactive. We are currently testing all mutants when expressed in bacteria (recombinant purified protein) or when transfected into mammalian cell lines (immunoprecipitated proteins). With these experiments, we hope to learn more about the regulation of the CDK6/KSHV-cyclin activity. Aim 3: In vivo functions of phospho-threonine 160 in Cdk2. The functions of phosphorylation of Thr160 in Cdk2 have been studied in yeast, mammalian cell lines, and using purified proteins. These experiments suggest that this phosphorylation is essential for activity of Cdk2. Recently, there have been indications that phosphorylation at Thr160 might not be absolutely essential in vivo. Therefore, we plan to knockin Cdk2T160A into the Cdk2 locus and study the effect of unphosphorylated Cdk2 in the mouse. In addition, we plan to screen for inhibitors of Cdk2 phosphorylation by an in vitro assay. In this assay, we monitor Cdk2 Thr160-phosphorylation by using our phospho-specific antibody. We have developed a high throughput screen on 96-well plates that needs to be adapted to robots. Once this is done, we will start to screen the natural product library and a defined chemical library. The combination of genetics and biochemistry/pharmacology will help us to understand the functions of Thr160 phosphorylation in vivo.